The present invention relates generally to methods and systems for examining biological materials and more particularly to methods and systems for examining biological materials using Raman spectroscopy.
A variety of Raman spectroscopic techniques have been reported in the art as being either useful or potentially useful in medical applications. For example, in U.S. Pat. No. 5,261,410, inventors Alfano et al., which issued Nov. 16, 1993, and which is incorporated herein by reference, there is disclosed a method for determining if a tissue is a malignant tumor tissue, a benign tumor tissue, or a normal or benign tissue using Raman spectroscopy. The method is based on the discovery that, when irradiated with a beam of infrared, monochromatic light, malignant tumor tissue, benign tumor tissue, and normal or benign tissue produce distinguishable Raman spectra. For human breast tissue, some salient differences in the respective Raman spectra are the presence of four Raman bands at a Raman shift of about 1078, 1300, 1445 and 1651 cm.sup.-1 for normal or benign tissue, the presence of three Raman bands at a Raman shift of about 1240, 1445 and 1659 cm.sup.-1 for benign tumor tissue, and the presence of two Raman bands at a Raman shift of about 1445 and 1651 cm.sup.-1 for malignant tumor tissue. In addition, it was discovered that, for human breast tissue, the ratio of intensities of the Raman bands at a Raman shift of about 1445 and 1650 cm.sup.-1 is about 1.25 for normal or benign tissue, about 0.93 for benign tumor tissue, and about 0.87 for malignant tumor tissue.
As another example, in U.S. Pat. No. 5,293,872, inventors Alfano et al., which issued Mar. 15, 1994, and which is incorporated herein by reference, there is disclosed a method for distinguishing between, on one hand, calcified atherosclerotic tissue and, on the other hand, fibrous atherosclerotic tissue or normal cardiovascular tissue using Raman spectroscopy. The method is based on the discovery that, when irradiated with a beam of monochromatic infrared light, calcified atherosclerotic human aortic tissue produces a Fourier Transform Raman spectrum which is distinguishable from analogous spectra obtained from fibrous atherosclerotic human aortic tissue and normal human aortic tissue. Some salient differences in the respective Raman spectra are the presence of five Raman bands at Raman shifts of 957, 1071, 1262-1300, 1445, and 1659 cm.sup.-1 (.+-.4 cm.sup.-2 for all shifts) for the calcified tissue as compared to three Raman bands at Raman shifts of 1247-1270, 1453 and 1659 cm.sup.-1 (.+-.4 cm.sup.-1 for all shifts) for the fibrous tissue and three Raman bands at Raman shifts of 1247-1270, 1449 and 1651 cm.sup.-1 (.+-.4 cm.sup.-1 for all shifts) for the normal tissue. In addition, it was discovered that the ratios of intensities for the Raman bands at 1659 and 1453 cm.sup.- and at 1254 and 1453 cm.sup.-1 were 0.69 and 0.53, respectively, for the calcified tissue, 1.02 and 0.85, respectively, for the fibrous tissue and 1.2 and 0.83, respectively, for the normal tissue.
As yet another example, in U.S. Pat. No. 5,243,983, inventors Tarr et al., which issued Sep. 14, 1993, and which is incorporated herein by reference, there is disclosed a non-invasive blood glucose measurement system and method using stimulated Raman spectroscopy. The system and method make use of two monochromatic laser beams, a pump laser beam and a probe laser beam. The output power of the pump laser beam is amplitude modulated, combined with the probe laser beam and directed into the ocular aqueous humor of a living being. The introduction of the laser beams into the ocular aqueous humor induces scattered Raman radiation, which causes a portion of the energy at the pump frequency to shift over to the probe frequency. The pump and probe laser beams are then detected as they exit the ocular aqueous humor. The probe laser beam is filtered, converted into an electrical signal and amplified. It is then compared to the modulation signal to generate an electrical signal representative of the concentration of D-glucose in the ocular aqueous humor.
Some of the problems with Raman spectroscopic techniques of the type described above (e.g., spontaneous Raman spectroscopy, coherent anti-stokes Raman spectroscopy and pulse-pumped stimulated Raman spectroscopy) are that such techniques typically require a long exposure time (i.e., several minutes) and high power laser pump fluency that exceeds the safety limitations for laser illumination in human body applications. As can readily be appreciated, requirements such as these substantially limit the practical applicability of these techniques in the fields of in vivo and in vitro medical diagnostics.
Other documents of interest include Owyoung, "Coherent Raman Gain Spectroscopy Using CW Laser Sources," IEEE Journal of Quantum Electronics, QE-14(3):192-202 (1978); Liu et al., "Raman, fluorescence, and time-resolved light scattering as optical diagnostic techniques to separate diseased and normal biomedical media, J. Photochem. Photobiol. B: Biol., 16:187-209 (1992); Lowenstein et al., "Nitric Oxide: A Physiologic Messenger," Ann Intern Med., 120:227-237 (1994); and Berger et al., "Feasibility of measuring blood glucose concentration by near-infrared Raman spectroscopy," Spectrochimica Acta Part A, 53:287-292 (1997), all of which are incorporated herein by reference.